An in Vitro System for Accurate Methylation of Internal Adenosine Residues in Messenger RNA

Narayan, Prema; Rottman, Fritz M.

doi:10.1126/science.3187541

Cited by 142 publications

(121 citation statements)

References 15 publications

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“…Among the sixteen sites tested, only four showed m 6 A modification >5% (Table 2). This result shows that the previously reported 20% m 6 A modification in bovine prolactin mRNA (Narayan and Rottman 1988) is not abnormal, suggests that many sites in mRNA carry incomplete m 6 A modification, and highlights that not every RRACH consensus sequence sites under the m 6 A/MeRIP-seq peaks are modified.…”

Section: Resultssupporting

confidence: 61%

Probing N⁶-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA

Liu

Parisien

Dai

et al. 2013

RNA

452

490

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N 6 -methyladenosine (m 6 A) is the most abundant modification in mammalian mRNA and long noncoding RNA (lncRNA). Recent discoveries of two m 6 A demethylases and cell-type and cell-state-dependent m 6 A patterns indicate that m 6 A modifications are highly dynamic and likely play important biological roles for RNA akin to DNA methylation or histone modification. Proposed functions for m 6 A modification include mRNA splicing, export, stability, and immune tolerance; but m 6 A studies have been hindered by the lack of methods for its identification at single nucleotide resolution. Here, we develop a method that accurately determines m 6 A status at any site in mRNA/lncRNA, termed site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET). The method determines the precise location of the m 6 A residue and its modification fraction, which are crucial parameters in probing the cellular dynamics of m 6 A modification. We applied the method to determine the m 6 A status at several sites in two human lncRNAs and three human mRNAs and found that m 6 A fraction varies between 6% and 80% among these sites. We also found that many m 6 A candidate sites in these RNAs are however not modified. The precise determination of m 6 A status in a long noncoding RNA also enables the identification of an m 6 A-containing RNA structural motif.

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Section: Resultssupporting

confidence: 61%

Probing N⁶-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA

Liu

Parisien

Dai

et al. 2013

RNA

452

490

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“…m 6 A is found within a conserved consensus sequence (G/A)(G/A) A CU (with a preference for (G/A)G A CU) in different eukaryotes (Horowitz et al ., 1984; Narayan & Rottman, 1988; Dominissini et al ., 2012; Meyer et al ., 2012; Luo et al ., 2014; Wan et al ., 2015). However, only a subset of potential sites is actually modified and the mechanisms underlying the specificity of writing are not yet understood.…”

Section: Introductionmentioning

confidence: 99%

Identification of factors required for m⁶A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI

et al. 2017

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Summary N6‐adenosine methylation (m6A) of mRNA is an essential process in most eukaryotes, but its role and the status of factors accompanying this modification are still poorly understood.Using combined methods of genetics, proteomics and RNA biochemistry, we identified a core set of mRNA m6A writer proteins in Arabidopsis thaliana.The components required for m6A in Arabidopsis included MTA, MTB, FIP37, VIRILIZER and the E3 ubiquitin ligase HAKAI. Downregulation of these proteins led to reduced relative m6A levels and shared pleiotropic phenotypes, which included aberrant vascular formation in the root, indicating that correct m6A methylation plays a role in developmental decisions during pattern formation.The conservation of these proteins amongst eukaryotes and the demonstration of a role in writing m6A for the E3 ubiquitin ligase HAKAI is likely to be of considerable relevance beyond the plant sciences.

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“…1A) had been bound (21). Following hybridization, filters were treated with RNase T1 to remove unhybridized RNA and washed and the RNA was eluted (22 (Du Pont Co.), and analyzed by fluorography as described previously (22). The exact sizes of the oligonucleotides generated were determined by comparison with an RNA ladder generated by alkaline hydrolysis of 3'-end-labeled RNA and analyzed in parallel (data not shown).…”

Section: Methodsmentioning

confidence: 99%

“…Unlabeled RNA (2 ,ug) was methylated by using S- [3H] adenosylmethionine in an in vitro methylation system consisting of a HeLa cell nuclear extract optimized for methyltransferase activity. This system has been shown previously to methylate accurately a single adenosine residue within the mature bPRL mRNA (22). Following incubation, the reaction mixture was treated with proteinase K, extracted with phenol-chloroform (1:1), and precipitated with ethanol.…”

Section: Methodsmentioning

confidence: 99%

“…More recent observations suggest a high frequency of methylation at PuGm6ACU sequences in Rous sarcoma virus RNA (18) and bovine prolactin (bPRL) mRNA (22). These data extend the core consensus sequence observed in mouse L cells (32), where in both heterogeneous nuclear RNA and mRNA about 65% of internal m6A occurs in the sequence PuGm6ACN, with the nucleotide N rarely being a G. The method of analysis, however, could not determine the relative frequencies of the other three bases at the N position.…”

mentioning

confidence: 99%

See 1 more Smart Citation

N6-methyladenosine residues in an intron-specific region of prolactin pre-mRNA.

Carroll¹,

Narayan²,

Rottman³

1990

Mol. Cell. Biol.

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N6-methyladenosine (m6A) residues occur at internal positions in most cellular and viral RNAs; both heterogeneous nuclear RNA and mRNA are involved. This modffication arises by enzymatic transfer of a methyl group from S-adenosylmethionine to the central adenosine residue in the canonical sequence G/AAC. Thus suggested following the precise localization of m6A in Rous sarcoma virus (18). In that molecule, the modified residues were found bracketing the src splice acceptor site and therefore may influence in some way the accuracy and/or efficiency of processing. Methylation of internal adenosine residues of eucaryotic cell RNA also takes place in the nucleus, as evidenced by the demonstration of m6A in eucaryotic cell heterogeneous nuclear RNA (24). However, previous analysis indicated a conservation of m6A residues during processing and transport to the cytoplasm, suggesting that this modification occurs mainly within exon regions (19). It was therefore of great interest to determine whether m6A occurs in intron-specific regions of eucaryotic precursor RNA. This analysis would identify the temporal relationship between transcription, methylation, and nuclear processing and may assist in developing a concept for the role of this posttranscriptional modification in eucaryotic RNA biogenesis.Attempts have been made to determine the influence of m6A on processing of gene transcripts. Earlier studies with cycloleucine, an inhibitor of S-adenosylmethionine synthetase, suggested that m6A affects the splicing of avian sarcoma virus genomic RNA (34) and processing of CHO cell mRNA (2). This hypothesis is supported by the similarity between the m6A consensus sequences in both eucaryotic and viral RNA and the weakly conserved branch point sequence YNCU(G/A)AC used in intron removal (18,27,40). Camper et al. (5) used the mRNA methylation inhibitor S-tubercidinyl homocysteine to measure the effects of undermethylation on the half-life and cytoplasmic appearance of poly(A)+ RNA in HeLa cells. A significant effect on processing and/or transport of undermethylated mRNA occurred with a delay in its appearance in the cytoplasm, whereas no significant alteration in the cytoplasmic half-life of undermethylated mRNA was observed.

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An in Vitro System for Accurate Methylation of Internal Adenosine Residues in Messenger RNA

Cited by 142 publications

References 15 publications

Probing N⁶-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA

Probing N⁶-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA

Identification of factors required for m⁶A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI

N6-methyladenosine residues in an intron-specific region of prolactin pre-mRNA.

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An in Vitro System for Accurate Methylation of Internal Adenosine Residues in Messenger RNA

Cited by 142 publications

References 15 publications

Probing N6-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA

Probing N6-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA

Identification of factors required for m6A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI

N6-methyladenosine residues in an intron-specific region of prolactin pre-mRNA.

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Resources

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Probing N⁶-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA

Probing N⁶-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA

Identification of factors required for m⁶A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI